Target Team: Jairo V, Saniya H, Stacy T, Daren Target (protein/gene name): Serine Threonine Phosphatase, STP1 SAK_0388 NCBI Gene #: 3686217 Protein ID (NP or XP #): YP_329040 Organism (including strain): Streptococcus Agalactiae, Strain A909 Etiologic Risk Group: Risk Group 2 Disease Information: Streptococcus Agalactiae is a bacterial infection that is mostly found in the intestinal tracts of humans, but it can also reside in the genital tracts of women. If a woman is infected with the bacteria, it can be transferred to the newborn baby through the amniotic fluid during childbirth. The newborn baby would then either develop the early or late onset disease which have the symptoms of respiratory distress, fever, lethargy, irritability, apnea and hypotension, but the late onset disease also causes meningitis and bacterimia. Only 2% of all the babies born are infected with this bacteria, and out all of those, more than 60% of the newborn babies become infected within the first 24 hours, and only 8% become infected after 2 days.The mortality rate, even with current treatments, exceeds 20%, and it becomes twice as deadly on older infants. It also affects people who are older than 65, with a 15% mortality rate, and adults who suffer alcoholism, diabetes, HIV, etc. Streptococcus Agalactiea is susceptible to combinations of ampicillin and penicillin,vancomycin, ciprofloxacin, clindamycin, erythromycin, cotrimoxazole, ceftriaxone, and it is resistant to macrolides and clindamycin. Image 1. Individual colony of Streptococcus Agalactiae.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): link to Sigma (or other company) page for assay (see Sigma links below) link (or citation) to paper that contains assay information links to assay reagents (substrates) pages. List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) PDB #: 2PK0 Ligands: Chloride Ions, Glycerol and Magnesium Ions.
-- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB Query Coverage: 100% Max % Identities: 99% % Positives: 244/245 (99%) Chain used for homology: A, B, C, D
Structure of the S. agalactiae serine/threonine phosphatase with protein chains colored from the N-terminal to the C-terminal.
Current Inhibitors: Okadaic Acid, Microcystin LR Cantharidin, Inhibitor-2, EDTA & FK-506. Expression Information (has it been expressed in bacterial cells): Purification Method: Image of protein: Image 2. The PyMol image of the STP1 protein, with the main chain carbons colored green and shown as ribbons, the magnesium ions shown as red spheres, the chlorine ions shown as yellow spheres, the glycerol shown as magenta and the active site cyan.
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): 1 ATGGAGATCAGCCTGCTCACTGACATTGGCCAGCGTCGTTCTAACAATCAGGACTTCATC 61 AATCAGTTCGAAAACAAAGCGGGTGTTCCGCTGATCATCCTGGCGGACGGCATGGGCGGT 121 CACCGTGCGGGCAACATCGCTTCCGAAATGACCGTTACCGACCTGGGTTCCGACTGGGCG 181 GAGACCGACTTCTCTGAACTGTCTGAAATTCGTGACTGGATGCTGGTATCTATCGAAACC 241 GAAAACCGTAAAATCTACGAACTCGGTCAATCTGACGACTACAAAGGTATGGGTACCACC 301 ATCGAAGCCGTAGCGATCGTTGGTGACAATATCATCTTCGCGCACGTCGGTGACTCTCGT 361 ATCGGTATCGTTCGTCAAGGTGAATATCACCTGCTGACGAGCGACCACTCTCTGGTTAAC 421 GAACTGGTCAAAGCAGGCCAGCTGACTGAAGAGGAAGCGGCGTCTCACCCGCAGAAAAAC 481 ATCATCACCCAGTCTATCGGTCAGGCGAATCCGGTTGAACCGGACCTCGGTGTACATCTG 541 CTGGAAGAAGGTGACTACCTGGTTGTTAACTCTGACGGTCTGACCAACATGCTGTCTAAC 601 GCGGACATCGCGACCGTTCTGACCCAGGAAAAACCGCTGGACGACAAAAACCAGGACCTG 661 ATCACCCTGGCGAACCATCGTGGTGGTCTGGACAACATCACCGTTGCGCTGGTTTACGTT 721 GAATCTGAAGCGGTATAA
GC% Content for gene (codon optimized): 52.8%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Target (protein/gene name): Serine Threonine Phosphatase, STP1 SAK_0388
NCBI Gene #: 3686217
Protein ID (NP or XP #): YP_329040
Organism (including strain): Streptococcus Agalactiae, Strain A909
Etiologic Risk Group: Risk Group 2
Disease Information: Streptococcus Agalactiae is a bacterial infection that is mostly found in the intestinal tracts of humans, but it can also reside in the genital tracts of women. If a woman is infected with the bacteria, it can be transferred to the newborn baby through the amniotic fluid during childbirth. The newborn baby would then either develop the early or late onset disease which have the symptoms of respiratory distress, fever, lethargy, irritability, apnea and hypotension, but the late onset disease also causes meningitis and bacterimia. Only 2% of all the babies born are infected with this bacteria, and out all of those, more than 60% of the newborn babies become infected within the first 24 hours, and only 8% become infected after 2 days.The mortality rate, even with current treatments, exceeds 20%, and it becomes twice as deadly on older infants. It also affects people who are older than 65, with a 15% mortality rate, and adults who suffer alcoholism, diabetes, HIV, etc. Streptococcus Agalactiea is susceptible to combinations of ampicillin and penicillin,vancomycin, ciprofloxacin, clindamycin, erythromycin, cotrimoxazole, ceftriaxone, and it is resistant to macrolides and clindamycin.
Image 1. Individual colony of Streptococcus Agalactiae.
Potential Physiological Targets: GST-Stk1 & PpaC (via: http://www.phosphatase.biochem.vt.edu/p3d2.cfm?extf=accession&extref=P3D0411151)
Link to Gene Database page: http://www.ncbi.nlm.nih.gov/gene/3686217
http://www.uniprot.org/uniprot/Q3K363
Is it a monomer or multimer as biological unit? (prediction): monomer
EC#: 3.1.3.16
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.16
FIgure 1. The Brenda enzyme mechanism schematic for the reaction of STP1, a phosphoprotein, and water.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
link to Sigma (or other company) page for assay (see Sigma links below)
link (or citation) to paper that contains assay information
links to assay reagents (substrates) pages.
List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
PDB #: 2PK0
Ligands: Chloride Ions, Glycerol and Magnesium Ions.
-- For Homology Model option:
Show pairwise alignment of your BLASTP search in NCBI against the PDB
Query Coverage: 100%
Max % Identities: 99%
% Positives: 244/245 (99%)
Chain used for homology: A, B, C, D
Current Inhibitors: Okadaic Acid, Microcystin LR Cantharidin, Inhibitor-2, EDTA & FK-506.
Expression Information (has it been expressed in bacterial cells):
Purification Method:
Image of protein:
Image 2. The PyMol image of the STP1 protein, with the main chain carbons colored green and shown as ribbons, the magnesium ions shown as red spheres, the chlorine ions shown as yellow spheres, the glycerol shown as magenta and the active site cyan.
Amino Acid Sequence:
1 MEISLLTDIGQRRSNNQDFINQFENKAGVPLIILADGMGGHRAGNIASEMTVTDLGSDWA
61 ETDFSELSEIRDWMLVSIETENRKIYELGQSDDYKGMGTTIEAVAIVGDNIIFAHVGDSR
121 IGIVRQGEYHLLTSDHSLVNELVKAGQLTEEEAASHPQKNIITQSIGQANPVEPDLGVHL
181 LEEGDYLVVNSDGLTNMLSNADIATVLTQEKPLDDKNQDLITLANHRGGLDNITVALVYV
241 ESEAVX
Length of your protein in Amino Acids: 246 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 26840 kDA
Molar Extinction coefficient of your protein at 280 nm wavelength: 18450 M-1cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html):
CDS Gene Sequence (paste as text only):
GC% Content for gene: 37.1%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGGAGATCAGCCTGCTCACTGACATTGGCCAGCGTCGTTCTAACAATCAGGACTTCATC
61 AATCAGTTCGAAAACAAAGCGGGTGTTCCGCTGATCATCCTGGCGGACGGCATGGGCGGT
121 CACCGTGCGGGCAACATCGCTTCCGAAATGACCGTTACCGACCTGGGTTCCGACTGGGCG
181 GAGACCGACTTCTCTGAACTGTCTGAAATTCGTGACTGGATGCTGGTATCTATCGAAACC
241 GAAAACCGTAAAATCTACGAACTCGGTCAATCTGACGACTACAAAGGTATGGGTACCACC
301 ATCGAAGCCGTAGCGATCGTTGGTGACAATATCATCTTCGCGCACGTCGGTGACTCTCGT
361 ATCGGTATCGTTCGTCAAGGTGAATATCACCTGCTGACGAGCGACCACTCTCTGGTTAAC
421 GAACTGGTCAAAGCAGGCCAGCTGACTGAAGAGGAAGCGGCGTCTCACCCGCAGAAAAAC
481 ATCATCACCCAGTCTATCGGTCAGGCGAATCCGGTTGAACCGGACCTCGGTGTACATCTG
541 CTGGAAGAAGGTGACTACCTGGTTGTTAACTCTGACGGTCTGACCAACATGCTGTCTAAC
601 GCGGACATCGCGACCGTTCTGACCCAGGAAAAACCGCTGGACGACAAAAACCAGGACCTG
661 ATCACCCTGGCGAACCATCGTGGTGGTCTGGACAACATCACCGTTGCGCTGGTTTACGTT
721 GAATCTGAAGCGGTATAA
GC% Content for gene (codon optimized): 52.8%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
https://docs.google.com/document/d/1ib30OT6lGE97Oa7ta5o1mlVMiuNLtMUCS9A8EqAKTPI/edit
Primer design results for 'tail' primers (this is just 2 sequences):